The broad goal of our work is to understand the molecular mechanisms used to control gene expression. Regulation of gene expression is essential for the proper differentiation and maintenance of distinct cell types in eukaryotes. A detailed understanding of transcriptional regulation is important for medicine because specific diseases, including cancer, can result from abnormalities in gene expression. The yeast Saccharomyces cerevisiae has proven to be a model eukaryotic organism for many types of studies, including gene regulation. Importantly, the transcription regulatory machinery is conserved between yeast and vertebrates, and insights gained from studies in yeast are generally universal. Studying the transcriptional regulation of the yeast HO gene has been very productive in terms of identifying important factors and novel regulatory paradigms that are conserved in metazoans. The yeast HO gene encodes an endonuclease with a complex pattern of regulation. Studies of HO regulation have historically been very productive, leading to the first identification of important regulatory molecules and paradigms. These include cell cycle regulated DNA-binding factors, components of the Swi/Snf, SAGA, and Sin3/Rpd3 complexes, regulation of nuclear entry by phosphorylation, and the Ash1 mRNA that is specifically transported subcellularly. The sequential binding of transcription factors and their recruitment of coactivators was first demonstrated at HO. Importantly, the regulatory paradigms that have been identified at the yeast HO gene are conserved in metazoans. This proposal is focused on three mechanistic questions about how chromatin works to regulate the yeast HO gene. The HO gene is tightly repressed by chromatin, including by the Tup1 repressor protein that blocks coactivator recruitment. Recent work shows that Tup1 is acetylated. The Gcn5 acetyltransferase is normally required for HO activation, but changing specific acetylatable lysine residues to glutamines allows the promoter to be activated in the absence of Gcn5. This suggests that Tup1 acetylation turns off the repression function, and may turn Tup1 into an activator. The second project pursues our preliminary evidence that gene activation involves a long range interaction between promoter elements, driven by bi-directional nucleosome eviction. The final project biochemically and genetically studies a histone modification that weakens the interactions between histones and DNA and thus reduces nucleosome stability. We have found that a lysine to glutamine substitution at lysine 56 of histone H3 suppresses defects in both HO expression and growth caused by histone chaperone mutations. Experiments are proposed to gain insights as to how chaperones and H3-K56 are critical in promoting transcriptional activation Defects in proper gene regulation are associated with diseases such as cancer. These studies are relevant because they will provide mechanistic insights as to how genes are regulated.